Project 2 represents a complementary approach to the one presented in Project 1 for examining genetic diversity in the microbiomes of obese and lean twin pairs. Whereas Project 1 investigates genomic and metabolomic properties of the whole gut microbial community, Project 2focuses on the gene complement of two prominent members of the gut microbiota, offering a genome-anchored view of the microbiome. The fermentative bacterium B. thetatiotaomicron and the methanogenic archaeon M. smithii have been shown in gnotobiotic mouse models to have a synergistic effect on energy harvest from the diet. Project 2 explores the genomic content of populations of these two microbial species obtained from lean and obese monozygotic twins. Our central hypothesis is that the microbiome of obese individuals has a greater capacity for energy harvest: thus, we postulate that there may be an enhancement of energy-acquisition functions in the genomes of these two microbial species obtained from obese individuals. The microbial genome is a dynamic entity, shaped by multiple forces that include gene loss and gain via lateral gene transfer (LGT). One implication of LGT is that no single genome sequence can describe a 'species'. To understand the nature of a microbial species we therefore must focus on the "pan-genome", the sum of core, vertically inherited genes and the variable, strain-specific genes. Pan-genome size can be vastly larger than the genome of any single isolate, increasing with every isolate sequenced. Wewill determine the extent of variability in the pan-genomes, both in terms of functional gene content and the discovery rate of new genes/isolate, of strains isolated from MZ twin pairs and their mothers and selected based on their core gene phylogenetic (tree-based) relationships. In this Project, we will use a combination of methods including culturing of isolates, comparative genome hybridization, genome amplification and large-scale DNA sequencing. Using data analysis methods outlined in Projects 1 and 3, we will assess the functional consequences of obesity- related pan-genome differences in terms of overall microbial gut metabolism, including inter-species synergisms. In addition, the data generated in Project 2will be used as a starting point for the investigations by the Knightlaboratory in Project 3 to trace the flow of genes in the gut microbiome.